True single-molecule-sensitive probes can provide the technological advances needed for real-time characterization of dynamic and highly heterogeneous cellular processes. To date, single molecule methods have been effective in revealing the environmental and mechanistic heterogeneity of biological systems in vitro;however observation of intracellular dynamics remains fundamentally limited by poor optical properties, biological incompatibility and unavailability, low sustainable emission rates, and poor photostabilities of potential single molecule fluorophores. We have assembled an outstanding team to create and optimize a new class of highly photostable, fluorogenic single molecule probes capable of very high sustained single molecule emission rates with essentially no experimentally relevant blinking. These few-atom Ag nanodots encapsulated in short ss-DNA strands emit in the low background near IR region with emission rates that uniquely enable the few msec frame rates necessary to capture dynamics of freely diffusing protein targets without from poisoning spatial resolution and signal/noise. These are the only monovalent species with sufficient photostability and sustained (i.e. essentially non-blinking) high emission rates, maintaining small overall size (as small as 3nm total hydrodynamic diameter). The 10-fold background reduction relative to visible excitation afforded by the 10-fold brighter near IR nanodot emitters, together reaches the 100-fold improvements necessary for true intracellular single molecule dynamics to be followed and characterized. Through three specific Aims, we will develop these robust ultrabright and ultrasmall nanodots into specific, cytosolically available, in vivo fluorogenic biological labels that are non-emissive until bound to target protein. In Aim I we will create and characterize fluorogenic near IR-emitting nanodot probes with intein and SNAP tags for in vivo conjugation, suitable for intracellular single molecule studies through microinjection. In Aim II we will attach membrane transport functionality and fully characterize the pathways and efficiencies of direct or indirect cytosolic uptake and endosomal escape. The fluorogenic probes will not contribute to background as they are designed to be emissive only upon conjugation to the target. These studies lead to Aim III in which thioredoxin dynamics in response to introduced oxidative stress preferentially transports Trx1 into the nucleus. An orthogonal two-color labeling scheme will be employed and microscopic rates characterizing stress-induced trafficking of Trx1 and Trx2 will be characterized on the single molecule level. Our long-term goal is the production and dissemination of sufficiently sensitive probes for generalized imaging of intracellular single molecule dynamics. A specific long-term goal is the oxidative stress-induced dynamics of thioredoxin in spherical (and therefore higher background) T-cells to more fully understand immune response. This toolbox of modular, small, highly emissive and photostable nanodots will be generally applicable to a wide range of systems, even in the presence of fast intracellular diffusion, and will be made available to the community through this project. Public Health Relevance: Heterogeneity and flexibility in biological processes confer the adaptability that is crucial to survival. These unsynchronized dynamics can only be visualized through the development of greatly improved protein labels that enable the unraveling of intracellular pathways through single molecule interactions. While being generally applicable to other intracellular dynamics studies, our development of multifunctional, modular Ag nanodots will lead to observation of synchronous multi-protein dynamics associated with redox regulation implicated in adaptive immune response and cancer.